Single molecule sequencing is used to obtain high-throughput sequence information on individual DNA or RNA. See, Braslaysky, Proc. Natl. Acad. Sci. USA 100: 3960-64 (2003). Sequencing-by-synthesis involves template-dependent addition of nucleotides to a template/primer duplex. Nucleotide addition is mediated by a polymerase enzyme and nucleotides may be labeled in order to facilitate their detection. All four Watson-Crick nucleotides may be added simultaneously, each with a different detectable label or nucleotides may be added one at a time in a step-and-repeat manner for imaging incorporation.
A challenge that has arisen in single molecule sequencing involves the ability to sequence through homopolymer regions (i.e., portions of the template that contain consecutive identical nucleotides). Often the number of bases present in a homopolymer region is important from the point of view of genetic function. As most polymerases used in sequencing-by-synthesis reactions are highly-processive, they tend to add bases continuously as the polymerase traverses a homopolymer region. Detectable labels used in sequencing generally have not been able to be resolved over multiple consecutive incorporations.
Some approaches have been devised that are intended to address the problem encountered with a highly-processive polymerase, but these approaches result in a nucleotide that is “scarred” upon release of inhibition. These “scarred” nucleotides often serve as poor substrates for the polymerase enzyme, making subsequent incorporations difficult. Thus, there is a tension between the desire to achieve step-and-repeat nucleotide incorporation and the desire for a highly-processive polymerase that will easily incorporate nucleotide analogs.
There is a need for detectably labeled nucleotides that do not hinder or inhibit ability of a polymerase to incorporate subsequent detectably labeled nucleotides into a nascent strand of DNA or RNA.